Nucleic acid-based testing is emerging as a promising method for detecting cancer and many other diseases. Retrieving highly purified nucleic acids from a clinical sample is an essential part of nucleic acid-based testing because it is based on analysis of the nucleic acid biomarker retrieved from the sample. In clinical specimen such as stool, the quantity of the nucleic acid biomarker is low and the biomarker may constitute only a small portion of total nucleic acids in a sample, meaning that detecting the rare biomarker requires the extraction of sufficient amounts of nucleic acids for enzymatic-based analysis. In other words, methods for nucleic acid extraction and purification must be able to effectively convert a large clinical sample to highly concentrated, highly purified nucleic acids so that enough amounts of the nucleic acid biomarker can be loaded for analysis.
Nucleic acid extraction and purification methods include alcohol precipitation, binding of nucleic acids to silica in the presence of chaotropic salts, gel filtration, ion exchange, and sequence-specific capture. However, when these methods are used alone, they are often not effective in preparation of highly concentrated, highly purified nucleic acids from clinical specimen, especially from a large clinical sample. This is because clinical specimen is highly complex and contains large amounts of interfering substances that can significantly reduce the effectiveness of nucleic acid extraction and purification methods, leading to a failure of recovering or detecting the nucleic acid biomarker from the clinical sample. For example, current methods are not effective in preparation of highly purified DNA from stool. Moreover, clinical sample contains a large amount of various inhibitors that could be co-isolated and concentrated with nucleic acids, inhibiting enzymatic-based analysis. As a result, their presence can limit the total quantity of nucleic acids that can be loaded for analysis because these inhibitors inhibit enzymatic-based analysis if their level is high in the system, leading to a failure of detecting the nucleic acid biomarker from the sample.
Pre-treating clinical specimen such as stool before nucleic acid extraction and purification is a means to reduce the effect of the interfering substances and inhibitors on extraction and detection of nucleic acids. The prior art pre-treatment methods include cetyl trimethylammonium bromide (CTAB) treatment, phenol/chloroform extraction, and protease treatment. These methods can be used either alone or in combination. However, these prior pre-treatment methods are not very effective when nucleic acids are extracted from a large clinical sample because of labor intensive protocols including multiple centrifugation and sample transfer steps.
Clearly, there is a need for methods that can effectively extract nucleic acids from a large clinical sample, while minimizing co-isolation of the inhibitors so that enough amounts of nucleic acids can be loaded (or used) for enzymatic-based analysis. The present invention addresses the issue associated with extraction and purification of nucleic acids from a sample containing nucleic acids, particularly from a large clinical sample.